CN110170071A - The method for promoting the degradation of alginic acid alkali 3D printing bio-ink inside and outside and cytochrome oxidase isozymes to stick - Google Patents

Abstract

The invention discloses a method for promoting the in vivo and in vitro degradation of alginate-based 3D printing bioink and cell stretching and adhesion, comprising: step 1, using alginate as the main skeleton of the extracellular matrix to construct a 3D bioprinting containing cells Bio-ink; step 2, mix alginate-based bio-ink with alginate lyase; step 3, add cell suspension to the obtained bio-ink, select printing parameters and print out 3D printing block through 3D bioprinter ; Step 4. According to the actual needs, adjust the degradation time of the 3D printing block in vitro and after transplantation and the degree of cell extension and adhesion by adjusting the concentration of alginate lyase. The method of the present invention can promote the degradation of the bio-ink with sodium alginate as the skeleton in vivo and in vitro and the stretching and adhesion of cells, so that the sodium alginate 3D printing bio-ink can adapt to more experiments in vivo and in vitro and better For soft tissue repair and transplantation.

Description

Promoting in vivo and in vitro degradation and cell extension adhesion of alginate-based 3D printing bioink Methods

technical field

The invention relates to a biological 3D printing and an application method thereof, in particular to a method for promoting the degradation of alginate-based 3D printing bio-ink in vivo and in vitro and cell stretching and adhesion.

Background technique

At present, the biggest limitation of organ transplantation is that the number of donor organs is very limited. Even autologous tissue cell transplantation, in the event of large-scale tissue damage (such as large-area deep skin burns, multiple tendon injuries, large blood vessel defects, etc.) Not enough organs for transplant are available. However, the use of exogenous materials as tissue substitutes, such as skin patches, dressings, dura mater patches, urethral patches, pelvic floor ligament suspension patches, etc., although they can play a temporary protective function, also involve biological aspects. Capacitance, in vivo degradative activity, cell stretch adhesion, chronic infection, and health economics are the limitations of many factors.

A good soft tissue substitute should not only be biocompatible, but also need to be degraded by the body within a certain period of time, so that the regenerated tissue of the body can fill and repair the defect. The difference between soft tissue defect and hard tissue defect, such as bone and cartilage defect, is that in addition to providing certain biomechanical support, soft tissue also has physiological, histological and other unique functions of living organisms, such as smooth muscle contraction function, skin secretion and shielding functions. Therefore, good soft tissue repair needs to mobilize endogenous cells, or provide a suitable environment and space for endogenous repair of body tissues. Therefore, for biomaterial substitutes for soft tissue defects, their in vivo and in vitro degradation and cell stretch adhesion are an important property.

In recent years, with the advancement and renewal of biomaterials and mechanical technology, cell-containing 3D bioprinting technology has become a hot topic and technology in the field. Based on the temperature, pressure or ultraviolet response effects of various biological materials, the seed cells can be mixed with liquid biological materials before printing, and the ideal three-dimensional structure can be printed out through a 3D bioprinter. Compared with traditional tissue engineering methods, the advantage of 3D bioprinting lies in the uniform mixing of seed cells and biomaterial scaffolds in advance, rather than waiting for cells in vivo and in vitro to grow and migrate slowly on the surface of biomaterial scaffolds. In addition, the organoid model constructed by 3D bioprinting provides cells with a highly biomimetic microenvironment. Researchers found that in the 3D microenvironment, the transcriptome and proteomics of cells are far better than those of two-dimensional plane culture. . Tissue intrinsic cells, such as dermal fibroblasts, keratinocytes, nerve cells, smooth muscle cells, and vascular endothelial cells, can grow normally and maintain their characteristic features and biological behaviors in the 3D bioprinting microenvironment. Stem cells, such as embryonic stem cells, induced pluripotent stem cells, mesenchymal stem cells, neural stem cells, epidermal stem cells, etc., can not only maintain their stemness in the microenvironment of 3D printing, but also retain the expression of specific markers in the state of stemness maintenance . In a specific 3D printing microenvironment, such as special mechanical pressure, or the presence of inducible proteins or small molecules, stem cells can differentiate in a specific direction. In addition, in the latest study, the 3D printed microenvironment can also recruit endogenous stem cells and immune cells in the body, and its paracrine and immunological characteristics are superior to traditional tissue engineering implants.

A key point in cell-containing 3D printing is bio-ink, which is in direct contact with cells, and its performance not only affects gel-forming ability, printability, rheology coefficient and maintenance state after printing, but also directly affects the biological behavior of cells. Therefore, the optimization and improvement of bio-inks has always been the focus and difficulty to be overcome in the field of materials science and tissue engineering in recent years. Among them, alginate has become one of the most commonly used components for cell-containing 3D printing at home and abroad because of its good biocompatibility and stability after cross-linking. Its various combinations with gelatin, chitosan, agarose, collagen and other thickening macromolecular materials are currently the most common bioinks. However, alginate has certain limitations. Because the main skeleton of alginate is composed of D-mannuronic acid aldehyde and L-glucuronic acid ester through glycosidic bonds, it cannot be degraded in higher organisms, and the same is true for the alginate molecular network after cross-linking with calcium ions. This has caused the long-term residual problem after the bio-ink using alginate as the main skeleton is transplanted into the body. For 3D bioprinting technology, this is a major obstacle to its future application and later promotion. In addition, alginate is a relatively inert biological material for cells, which lacks attachment sites for cell adhesion and migration. Cells in alginate hydrogels are mainly spherical, lacking the spindle-shaped, round shape that cells themselves should have. or other polygonal shapes. Because the geometric shape of cells has been shown to have a certain impact on the transcription level of cells, this performance of alginate has certain limitations for cells.

Contents of the invention

The purpose of the present invention is to provide a method of using bio-3D printing technology to promote the degradation of printing blocks in vivo and in vitro and cell stretching and adhesion, which can enhance or improve the in vivo and in vitro degradation and cell degradation of 3D printing inks with alginate as the main skeleton. Stretching adhesion can solve biological problems such as the degradation of printed blocks in vivo after printing with alginate-based bioinks, and promote cell release, stretching and migration.

In order to achieve the above purpose, the present invention provides a method for promoting the degradation of alginate-based 3D printing bioink in vivo and in vitro and cell extension and adhesion, wherein the method includes: step 1, using alginate as the extracellular matrix Representative extracellular matrix components of the main body skeleton Biomaterials, such as sodium alginate and gelatin, are used as extracellular matrix to construct 3D bioprinted bioinks containing cells; step 2, with different concentrations of alginate lyase and alginate base Bio-ink, such as sodium alginate-gelatin bio-ink mixing; step 3, adding cell suspension to the bio-ink obtained in step 2, after selecting certain printing parameters, including mechanical parameters, software parameters, etc., through extrusion 3D The bioprinter prints a 3D printing block that can be degraded under control, that is, a 3D printing model; step 4, according to the actual needs of 3D printing and the specific needs of the experiment, adjust the concentration of alginate lyase to regulate the cultivation and transplantation of the 3D printing block in vitro In vivo post-degradation time and extent of cell stretch adhesion.

The above-mentioned method for promoting the degradation of alginate-based 3D printing bioink in vivo and in vitro and cell extension and adhesion, wherein the cells in the bioink containing 3D bioprinting containing cells include tissue intrinsic cells and stem cells with the ability to proliferate and differentiate and stem cells with functions of paracrine secretion, vesicle secretion and exosome release; the tissue intrinsic cells include fibroblasts, epithelial keratinocytes, neuronal cells, glial cells, vascular endothelial cells, chondrocytes, osteoblasts Any one or more of cells, muscle cells, small intestinal epithelial cells, etc.; the stem cells with proliferation and differentiation and the stem cells with paracrine, vesicle secretion and exosome release functions include embryonic stem cells, induced multiple Any one or more of competent stem cells, mesenchymal stem cells, epidermal stem cells, neural stem cells, muscle stem cells, small intestinal crypt cells, etc.

The above-mentioned method for promoting the degradation of alginate-based 3D printing bioink in vivo and in vitro and cell stretching and adhesion, wherein the representative extracellular matrix component biomaterial, that is, alginate-based bioink, is based on alginate The cross-linked skeleton, as the main macromolecule to maintain the shape of the printed block after printing, also contains A-type gelatin, B-type gelatin, chitosan, hyaluronic acid, collagen (including type I collagen and type IV collagen), agarose, A mixture of any one or more of fibrin, silk protein, and synthetic biological materials, such as PLGA (poly lactic-co-glycolic acid, polylactic-co-glycolic acid copolymer), etc., and alginate.

The above-mentioned method for promoting the degradation of alginate-based 3D printing bio-inks in vivo and in vitro and cell stretching and adhesion, wherein the printing parameters include printing nozzle aperture, printing nozzle moving speed, printing distance, printing layer thickness, printing air pressure, cross-linking agent type, crosslinking agent concentration, crosslinking temperature and time, etc.

The above-mentioned method for promoting the degradation of alginate-based 3D printing bioink in vivo and in vitro and cell extension and adhesion, wherein the step 2 includes: step 2.1, after weighing the powder of alginate lyase, use deionized water to configure 100mU/ml mother solution, store in 4°C refrigerator for use; step 2.2, put 10ml of bio-ink, such as sodium alginate/gelatin bio-ink, at a temperature of 37°C, preferably in a 37°C water bath. After the bio-ink is completely melted into a liquid state, add the alginate lyase mother solution prepared in step 2.1, and turn it upside down to mix well and gently to prevent a large number of small air bubbles from being mixed.

The above-mentioned method for promoting the degradation of alginate-based 3D printing bioink in vivo and in vitro, wherein, in the step 2.1, the source species of alginate lyase include marine algae, marine molluscs, echinoderms, marine bacteria, soil bacteria Any one or more of fungi, etc.; the molecular weight of the enzyme is 24-100kDa, and the optimum pH is between 6.0-11.0. The enzyme cleaves 4-O-glycosidic bonds via a mechanism of β-elimination of β-1,4-glycosidic bonds between monomers. After cleavage, an oligomeric uronic acid containing 4,5-unsaturated double bonds is generated on the non-reducing terminal C ₄ and C ₅ of the product. This unit has a strong ultraviolet absorption at 230-240nm, so it is the key to the enzyme activity. test methods. The specific method for measuring the enzyme activity is: add 0.15ml (1un/ml) alginate lyase solution to 0.1% (w/v) sodium alginate solution (pH6.3) and incubate. One unit of enzyme activity is defined as an increase in A _235nm of 1.0 per minute at 37°C and pH 6.3 per milliliter of sodium alginate substrate.

The above-mentioned method for promoting the degradation of alginate-based 3D printing bio-ink in vivo and in vitro, wherein, in the step 2.2, the centrifuge tube containing the bio-ink is completely immersed in a water bath at 37°C for a time greater than or equal to 1 hour, The bio-ink was completely melted into a liquid state, and then at this temperature, according to the specific experimental requirements, 5-50 μl of alginate lyase mother solution was added to the bio-ink, and mixed well, with gentle movements to avoid a large number of bubbles.

The above-mentioned method for promoting the degradation of alginate-based 3D printing bio-ink in vivo and in vitro, wherein the step 3 includes: step 3.1, adding 0.5ml of the cell suspension to the bio-ink prepared in step 2.2, fully mixing, and then Transfer it into a 3D bioprinted printing cylinder, push the bottom piston to the highest position, discharge the residual gas in the printing cylinder, tighten the printing nozzle piston, and seal it with a sealing film; the number of cells contained in 0.5ml of cell suspension is greater than or equal to 0.5× 10 ⁷ pieces; step 3.2, put the printing cylinder containing the bio-ink into a cold water bath and let it stand still, and when the bio-ink turns into a gel, use a nozzle with a specific aperture to print; step 3.3, select the printing parameter as the platform of the 3D bioprinter The temperature is 10°C, the temperature of the printing cylinder is 15°C, the printing speed is 10mm/s, and the pressure is 70-100kPa; step 3.4, cross-link the printed 3D printing block with CaCl ₂ solution; step 3.5, absorb it after cross-linking Remove residual CaCl ₂ solution, wash twice with complete medium, enough complete medium until the 3D printing block is covered, put in the incubator for one hour, change the medium again to completely remove residual calcium ions, and then long-term nourish.

The above-mentioned method for promoting the in vivo and in vitro degradation of alginate-based 3D printing bio-ink and cell stretching and adhesion, wherein, in the step 3.2, the printing cylinder containing the bio-ink is placed in a 15°C water bath for 15 minutes, and the biological When the ink turns into a gel, print with a nozzle with a diameter of 260 μm.

The above-mentioned method for promoting the degradation of alginate-based 3D printing bioink in vivo and in vitro and cell extension and adhesion, wherein, in the step 3.4, add 10ml of 2% (w/v) CaCl ₂ solution to the printed block at 37°C as an exchange Linking agent, make the solution submerge the printed block, and cross-link at room temperature for 10 minutes.

The method provided by the present invention to promote the degradation of alginate-based 3D printing bioink in vivo and in vitro has the following advantages:

The invention adopts the alginate lyase to adjust the degradation time of the alginate-based bio-ink after in vitro cultivation and in vivo transplantation. Alginate is used as the main skeleton molecule, and one or more macromolecules (including type A gelatin, type B gelatin, chitosan, hyaluronic acid, type I collagen, type IV collagen, fibrin, agarose, silk protein and synthetic biomaterials) are mixed to form bioinks. The network formed by the cross-linking of alginate and calcium ions can maintain the shape after printing. The concentration of alginate lyase can adjust the degradation time of the bioink in vitro and in vivo after printing, and at the same time, it does not affect the printing process, nor does it affect cell viability, and has no effect on cell proliferation, migration, adhesion and related protein expression. obvious impact. Under different printing conditions, according to the requirements of post-printing culture, cell release or degradation, different concentrations of sodium alginate lyase are set to print 3D bioprinted blocks with controllable degradation.

Cell-containing bioprinting has always had the problem that cells cannot stretch, adhere, and migrate well in hydrogels. The cells in the print block cannot reproduce the state of the cells in vivo with high fidelity. While controlling the degradation of the alginate-based bio-ink in vivo and in vitro, the invention changes the porosity and compressive modulus of the bio-ink, and promotes the adhesion, extension and migration of cells in the printing block. It provides a good method for the optimization of bioink for cell-containing 3D bioprinting. The research and application of biological 3D printing has developed rapidly in recent years, and the bio-ink with alginate as the main skeleton is the most commonly used one at home and abroad. The invention is still the first at home and abroad, and fills the gap in the optimization of alginate bio-ink degradation. In addition, although the effect of 3D microstructure changes on cell adhesion, extension and migration has been reported earlier, this invention proves that suitable 3D printed microstructures are more conducive to cells in vitro to exhibit a highly biomimetic in vivo state. From the perspective of space-time controllability and cost feasibility, this achievement optimizes the bio-ink based on alginate, and provides ideas for the later application, transformation, in-depth research and further optimization of this bio-ink in 3D bioprinted soft tissue and programs.

Description of drawings

Fig. 1 is a schematic diagram of the process of promoting the degradation of alginate-based 3D printing bio-ink in vivo and in vitro and cell extension and adhesion of the present invention.

Fig. 2 shows the degradation of printed blocks in vitro after printing with sodium alginate/gelatin bio-ink containing alginate lyase in an embodiment of the present invention.

Among them, Fig. 2A is three kinds of alginate lyase concentrations (0mU/ml, 0.5mU/ml, 5mU/ml) of sodium alginate/gelatin bioink printing blocks cultured in vitro for 3 days (D3), 7 days (D7) and the state after 14 days (D14). Figure 2B shows the H&E staining (hematoxylin, hematoxylin and eosin) of sodium alginate/gelatin bioink print blocks with three alginate lyase concentrations (0mU/ml, 0.5mU/ml, 5mU/ml) cultured in vitro for 3 days eosin staining) results.

Fig. 3 shows the in vivo degradation and cell retention of sodium alginate/gelatin bioinks containing different concentrations of alginate lyase in an embodiment of the present invention.

Among them, Figure 3A is a schematic diagram of H&E staining of tissue sections 7 days after subcutaneous transplantation of bio-ink printed blocks with different concentrations of alginate lyase. Figure 3B is an imaging image of living small animals after in vivo transplantation of cells with DiI fluorescent signals in printed blocks of alginate lyase at different concentrations. Fig. 3C is a line graph of fluorescence signal intensity quantification in Fig. 3B.

Fig. 4 shows the state of cells stretched in the bio-ink after printing with sodium alginate/gelatin bio-ink containing alginate lyase in an embodiment of the present invention.

Among them, Fig. 4A is the H&E staining high-magnification result of three alginate lyase concentrations (0mU/ml, 0.5mU/ml, 5mU/ml) of sodium alginate/gelatin bioink printing blocks cultured in vitro for 3 days. In the center of the picture are fibroblasts stretched in different bioinks. Fig. 4B shows that fibroblasts with GFP fluorescent protein were cultured in vitro for 3 days in sodium alginate/gelatin bioink printing blocks of three alginate lyase concentrations (0mU/ml, 0.5mU/ml, 5mU/ml), Status after 7 and 14 days. Figure 4C is a quantitative graph of the proportion of stretched cells to total cells after cells were cultured in three bioinks for 3 days, 7 days and 14 days. Figure 4D shows the vertical/horizontal axis ratios of stretched cells cultured in three bioinks for 3 days, 7 days and 14 days.

Detailed ways

Specific embodiments of the present invention will be further described below.

As shown in Figure 1, the method provided by the present invention to promote the degradation of alginate-based 3D printing bio-ink in vivo and in vitro and cell extension adhesion, the method includes:

Step 1. Using alginate as the main skeleton of the extracellular matrix as a representative extracellular matrix component biomaterial, such as sodium alginate and gelatin as the extracellular matrix, to construct a 3D bioprinted bioink containing cells. Using alginate as the main bio-ink skeleton, alginate forms a stable molecular network in the calcium ion cross-linking agent, which can maintain the shape of the printed block after printing and has good biocompatibility.

The cells in the 3D bioprinted bioink containing cells include tissue-intrinsic cells, stem cells with the ability to proliferate and differentiate, and stem cells with paracrine, vesicle-secreting, and exosome-releasing functions; the tissue-intrinsic cells include fibroblasts cells, epithelial keratinocytes, neuronal cells, glial cells, vascular endothelial cells, chondrocytes, osteoblasts, muscle cells, small intestinal epithelial cells, etc.; Stem cells and stem cells with paracrine, secretory vesicle and exosome functions, including any one of embryonic stem cells, induced pluripotent stem cells, mesenchymal stem cells, epidermal stem cells, neural stem cells, muscle stem cells, and small intestinal crypt cells one or more species.

Representative extracellular matrix component biomaterials, that is, alginate-based bioinks, use alginate as the main cross-linked skeleton as the main macromolecule to maintain the shape of the printed block after printing. In addition, it also contains A-type gelatin and B-type gelatin , chitosan, hyaluronic acid, collagen (including type I collagen, type IV collagen), agarose, fibrin, silk protein and synthetic biological materials, such as any one or more of PLGA and alginate mixture.

Step 2, mixing alginate-lyase with different concentrations of alginate-based bio-ink, such as sodium alginate-gelatin bio-ink.

Step 2 includes: Step 2.1. After weighing the powder of alginate lyase, use deionized water to prepare a mother solution of 100mU/ml, and store it in a refrigerator at 4°C until use.

Preferably, the source of alginate lyase includes any one or more of marine algae, marine molluscs, echinoderms, marine bacteria, soil bacteria and fungi; the molecular weight of the enzyme is 24-100 kDa, and the optimum pH is at Between 6.0 and 11.0. The enzyme cleaves 4-O-glycosidic bonds via a mechanism of β-elimination of β-1,4-glycosidic bonds between monomers. After cleavage, an oligomeric uronic acid containing 4,5-unsaturated double bonds is generated on the non-reducing terminal C ₄ and C ₅ of the product. This unit has a strong ultraviolet absorption at 230-240nm, so it is the key to the enzyme activity. test methods. The specific method for measuring the enzyme activity is: add 0.15ml (1un/ml) alginate lyase solution to 0.1% (w/v) sodium alginate solution (pH6.3) and incubate. One unit of enzyme activity is defined as an increase in A _235nm of 1.0 per minute at 37°C and pH 6.3 per milliliter of sodium alginate substrate.

Step 2.2. Put 10ml of bio-ink, such as sodium alginate/gelatin bio-ink, at a temperature of 37°C, preferably in a water bath at 37°C. After the bio-ink is completely melted and becomes liquid, add it to step 2.1 and configure Alginate lyase mother solution, and turn it upside down, mix well, and move gently to prevent mixing a large number of small air bubbles.

Preferably, the centrifuge tube containing the bio-ink is completely immersed in a water bath at 37°C for more than or equal to 1 hour, so that the bio-ink is completely melted into a liquid state, and then under this temperature condition, according to specific experimental requirements, 5 ~ Add 50 μl alginate lyase mother solution to the bio-ink, and mix well, with gentle movements to avoid a large number of air bubbles.

Step 3. Add cell suspension to the bio-ink obtained in step 2. After selecting certain printing parameters, including mechanical parameters, software parameters, etc., print a 3D printing block with controllable degradation through the extrusion 3D bioprinter. 3D printed models. Printing parameters include print head aperture, print head moving speed, print pitch, print layer thickness, print air pressure, type of cross-linking agent, concentration of cross-linking agent used, cross-linking temperature and time, etc.

Step 3 includes: step 3.1, adding 0.5ml of cell suspension to the bio-ink prepared in step 2.2, mixing thoroughly, and then transferring it into the 3D bioprinting cartridge, pushing the bottom piston to the highest position, and discharging the residue in the printing cartridge Gas, tighten the piston of the printing nozzle, and seal it with parafilm; the number of cells contained in 0.5ml of cell suspension is greater than or equal to 0.5×10 ⁷ .

Step 3.2. Put the printing cylinder containing the bio-ink into a cold water bath and let it stand still. When the bio-ink turns into a gel, print with a nozzle with a specific aperture. Preferably, put the printing cylinder containing the bio-ink into a 15°C water bath and let it stand for 15 minutes. When the bio-ink turns into a gel, print with a nozzle with a diameter of 260 μm.

Step 3.3. Select the printing parameters as the temperature of the platform of the 3D bioprinter is 10°C, the temperature of the printing cylinder is 15°C, the printing speed is 10mm/s, and the pressure is 70-100kPa.

Step 3.4, use CaCl ₂ solution to cross-link the printed 3D printing block; Step 3.5, absorb the residual CaCl ₂ solution after cross-linking, wash twice with complete medium, enough complete medium until no 3D printing After one hour in the incubator, change the medium again to completely remove residual calcium ions, and then cultivate for a long time. Preferably, 10 ml of 2% (w/v) CaCl ₂ solution is added to the printed block at 37° C. as a cross-linking agent, the solution is submerged in the printed block, and cross-linked at room temperature for 10 min.

Step 4. According to the actual needs of 3D printing and the specific needs of the experiment, adjust the concentration of alginate lyase to regulate the degradation time and cell extension and adhesion degree of the 3D printing block after in vitro culture and transplantation in vivo.

The method for promoting the degradation of alginate-based 3D printing bio-ink in vivo and in vitro and cell extension and adhesion provided by the present invention will be further described in conjunction with the examples below.

Example 1: Using alginate lyase to promote the in vivo and in vitro degradation of alginate-based bio-3D printing blocks.

Using alginate lyase mixed with alginate/gelatin bioink, as shown in Figure 2 and Figure 3, Figure 2A can be observed in vitro culture of printed blocks, the degradation time is related to the concentration of alginate lyase. Figure 2B shows the printed blocks of different concentrations of sodium alginate lyase, H&E staining results, the shaded part is alginate, and the degree of fragmentation of the alginate skeleton is significantly different with the change of enzyme concentration (scale bar: 200 μm). As shown in Figure 3B, the mouse dermal fibroblasts with DiI fluorescent signal were mixed with bio-ink, and the printed block was implanted subcutaneously in nude mice, and traced detection was performed using a living small animal instrument. As shown in Figure 3A, it is the result of sampling tissue sections after the print blocks were implanted subcutaneously in nude mice. H&E results showed that the degradation degree of print blocks containing different enzyme concentrations in nude mice had a significant correlation with the enzyme concentration. Blocks are alginate (scale bar: 200 μm).

The specific printing and cultivation process is as follows:

(1) Weigh and prepare 10 ml of an aqueous solution of 1% (w/v) sodium alginate and 3% (w/v) type B gelatin. (2) Pasteurization was used to sterilize the mixture of sodium alginate and gelatin, placed in a water bath at 70°C for 30 minutes, and then placed in a refrigerator at 4°C for 5 minutes. This process was repeated three times.

(3) Prepare the alginate lyase powder with deionized water to prepare a storage solution of 1000 mU/ml, and store it in a refrigerator at 4°C until use.

(4) The prepared bio-ink was taken out of the refrigerator, and placed in a water bath at 37° C. for 1 hour, so that the bio-ink was completely melted and became liquid.

(5) Take 50 μl of alginate lyase storage solution and add it to 10 ml of sodium alginate/gelatin bio-ink that has melted into a liquid state, turn it upside down, and mix well. The bioink contains alginate lyase at a final concentration of 5mU/ml.

(6) Digest and centrifuge adherent mouse dermal fibroblasts in three 100mm culture dishes with a density of 80-90% to prepare a cell suspension with a cell density of ¹ ×107 cells/ml. The volume is 0.5ml.

(7) Add the cell suspension to the bio-ink prepared above, use a pipette gun to blow and mix well, put the cell-containing bio-ink into the 3D printing cartridge, push the bottom piston to the highest position, and discharge the residual gas in the printing barrel , tighten the print nozzle piston, and seal it with parafilm.

(8) Put the printing cylinder in a cold water bath at 15°C and let it stand for 15 minutes, and start printing after the bio-ink turns into gel.

(9) Remove the sealing film at the printing nozzle, unscrew the piston, replace it with a nozzle with a diameter of 260 μm, and place the printing cylinder on the 3D printer.

(10) Turn on the power of the 3D printer and connect it to the computer, open the software, select the printing model parameters, the cylindrical shape is 3×3×0.5cm, the temperature of the printing platform is 10°C, the temperature of the printing cylinder is 15°C, and the printing speed is 10mm/s. The air pressure is 70-100kPa.

(11) Add 2% (w/v) CaCl ₂ solution to the printed block for cross-linking, the solution completely submerges the printed block, and cross-link at room temperature for 10 min.

(12) Aspirate the CaCl ₂ solution, wash twice with the medium, add the medium and put it in the incubator for 1 hour and then replace the medium again.

Example 2: Using alginate lyase to promote cell expansion and migration in alginate-based bio-3D printing blocks.

Under physiological conditions, cells are generally polygonal and have the characteristics of stretching and adhesion. Conventional alginate lacks cell attachment sites, and although it has good biocompatibility, cells stretch less in alginate print blocks. Using alginate lyase, as shown in Figure 4, can significantly increase cell stretching in the printed block. As shown in Figure 4A, in the print block without adding alginate lyase, the cells were mostly oval, and in the print block with alginate lyase, the formation of cell pseudopodia and cell stretching could be observed (scale bar: 50μm). Figure 4B shows the state of GFP fluorescent mouse dermal fibroblasts in alginate-based bioink. In the printing block added with alginate lyase, the proportion of cell extension to the total cells was significantly increased, and the ratio of cells on the vertical axis/horizontal axis also increased. Significant rise (scale bar: 50 μm). Figure 4C is a quantification graph of stretched cell ratio and cell vertical/horizontal axis ratio.

The method provided by the present invention to promote the in vivo and in vitro degradation of alginate-based 3D printing bioinks and cell extension and adhesion includes two parts: 1), using alginate lyase to promote the in vivo and in vitro degradation of alginate-based bio-3D printing blocks ; 2), using alginate lyase to promote cell extension and migration in alginate-based bio-3D printing blocks. The purpose is to promote the performance of alginate-based 3D printing inks, and guide new directions for the further upgrading, optimization and reform of related systems of bio-inks, so that they can be better applied to soft tissue construction in vitro, tissue repair and regeneration.

Although the content of the present invention has been described in detail through the above preferred embodiments, it should be understood that the above description should not be considered as limiting the present invention. Various modifications and alterations to the present invention will become apparent to those skilled in the art upon reading the above disclosure. Therefore, the protection scope of the present invention should be defined by the appended claims.

Claims (10)

1. A method for promoting alginate-based 3D printing bioink in vitro and in vivo degradation and cell extension adhesion, characterized in that, the method comprises: Step 1. Using alginate as a representative extracellular matrix component biomaterial as the main skeleton of the extracellular matrix to construct a 3D bioprinted bioink containing cells; Step 2, mixing alginate-lyase with alginate-based bio-ink; Step 3, adding the cell suspension to the bio-ink obtained in step 2, selecting the printing parameters, and printing out a 3D printing block with controllable degradation through the extrusion 3D bioprinter; Step 4. According to the actual needs of 3D printing, adjust the concentration of alginate lyase to regulate the degradation time of 3D printed blocks in vitro and after transplantation and the degree of cell extension and adhesion. 2. The method for promoting alginate-based 3D printing bioink in vitro and in vivo degradation and cell extension and adhesion as claimed in claim 1, wherein the cells in the bioink of the 3D bioprinting containing cells contain tissue intrinsic Cells, and stem cells with proliferation and differentiation and stem cells with paracrine, vesicle secretion and exosome release functions; the tissue intrinsic cells include fibroblasts, epithelial keratinocytes, neuron cells, glial cells, Any one or more of vascular endothelial cells, chondrocytes, osteoblasts, muscle cells, and small intestinal epithelial cells; the stem cells with proliferation and differentiation functions and the stem cells with paracrine, secretory vesicle, and exosome release functions Stem cells include any one or more of embryonic stem cells, induced pluripotent stem cells, mesenchymal stem cells, epidermal stem cells, neural stem cells, muscle stem cells, and small intestinal crypt cells. 3. The method for promoting the degradation of alginate-based 3D printing bio-inks in vivo and in vitro and cell extension and adhesion as claimed in claim 1, wherein the representative extracellular matrix component biomaterial is mainly composed of alginate The skeleton also includes a mixture of any one or more of A-type gelatin, B-type gelatin, chitosan, hyaluronic acid, collagen, agarose and synthetic biological material PLGA with alginate. 4. The method for promoting the degradation of alginate-based 3D printing bio-inks in vivo and in vitro and cell stretching and adhesion as claimed in claim 1, wherein the printing parameters include printing nozzle aperture, printing nozzle moving speed, printing distance, Printing layer thickness, printing air pressure, type of crosslinking agent, concentration of crosslinking agent used, as well as crosslinking temperature and time. 5. The method for promoting alginate-based 3D printing bio-ink in vitro and in vivo degradation and cell extension adhesion as claimed in claim 1, wherein said step 2 comprises: Step 2.1. Prepare the powder of alginate lyase with deionized water to prepare a mother solution of 1000mU/ml, and store it in a refrigerator at 4°C for later use; Step 2.2. After 10ml of bio-ink is completely melted at 37°C and becomes liquid, add the alginate lyase mother solution prepared in step 2.1 and mix well. 6. The method for promoting the degradation of alginate-based 3D printing bio-inks in vivo and in vitro and cell extension and adhesion as claimed in claim 5, characterized in that, in the step 2.1, the sources of alginate lyase include marine algae, Any one or more of marine molluscs, echinoderms, marine bacteria, soil bacteria and fungi; the molecular weight of the enzyme is 24-100kDa, and the optimum pH is between 6.0-11.0. 7. The method for promoting alginate-based 3D printing bio-ink degradation in vivo and in vitro and cell extension and adhesion as claimed in claim 5, characterized in that, in the step 2.2, the centrifuge tube containing the bio-ink is completely immersed in 37 In a water bath at ℃, the time is greater than or equal to 1 hour, so that the bio-ink is completely melted into a liquid state, and then 5-50 μl of alginate lyase mother solution is added to the bio-ink under this temperature condition, and mixed well. 8. The method for promoting alginate-based 3D printing bio-ink in vitro and in vivo degradation and cell extension adhesion as claimed in claim 1, wherein said step 3 comprises: Step 3.1. Add 0.5ml of the cell suspension to the bio-ink prepared in step 2.2 and mix well, then transfer it into the printing cylinder of the 3D bioprinter, tighten the piston of the printing nozzle, and seal it with a sealing film; the cells contained in the 0.5ml of the cell suspension The number is greater than or equal to 0.5× ¹⁰⁷ ; Step 3.2, put the printing cylinder in a cold water bath and let it stand, and when the bio-ink turns into a gel, print with the nozzle; Step 3.3, select the printing parameters as follows: the temperature of the platform of the 3D bioprinter is 10°C, the temperature of the printing cylinder is 15°C, the printing speed is 10mm/s, and the pressure is 70kPa～100kPa; Step 3.4, cross-linking the printed 3D printing block with CaCl ₂ solution; Step 3.5. After cross-linking, suck off the residual CaCl ₂ , wash twice with complete medium, add enough complete medium until the 3D printing block is covered, put it in the incubator for one hour, and change the medium again to remove Residual calcium ions, then long-term culture. 9. The method for promoting alginate-based 3D printing bio-ink degradation in vivo and in vitro and cell stretching and adhesion as claimed in claim 8, characterized in that, in step 3.2, the printing cylinder is placed in 15°C water for 15 minutes , when the bio-ink was transformed into a gel, it was printed with a nozzle with a diameter of 260 μm. 10. The method for promoting the degradation of alginate-based 3D printing bio-ink in vivo and in vitro and cell extension and adhesion as claimed in claim 8, characterized in that, in the step 3.4, 10ml of 2% is added to the printed 3D printing block The w/v CaCl ₂ solution was used as a cross-linking agent, and the solution was immersed in the printed block, and cross-linked at room temperature for 10 min.